Nucleic acid amplification techniques, including PCR and LAMP methods, have come to be used in many fields of biology such as molecular biology and medicine, and they are commonly used for DNA profiling, food tests, environmental sanitation tests and animal and plant tests.
When the test sample is a liquid it may be directly used as the nucleic acid amplification sample for amplification of the target nucleic acid, but since target nucleic acid is present in cells in the case of a biological sample, the biological sample must be dissolved by using a cell lysate (for example, an alkali solution or surfactant) and the nucleic acid eluted out of the cells to prepare the nucleic acid amplification sample.
For example, a solution containing sodium dodecylsulfate may be added to the biological sample and the mixture heated to denature the protein, or an aqueous sodium hydroxide solution added thereto, to disrupt the cell membranes and elute the nucleic acid for use as the nucleic acid amplification sample.
Expectorates and saliva are very important biological samples for diagnosis of lung disease such as tuberculosis.
However, it is usually difficult to stably amplify nucleic acid from expectorate or saliva, and therefore detection of Mycobacterium in expectorate or saliva for diagnosis is usually accomplished by liquefying the sampled expectorate or saliva with sodium hydroxide or the like and performing centrifugal separation, collecting the Mycobacterium cells as the precipitate, and culturing them (Patent document 1).
When preparing a nucleic acid amplification sample from a biological sample other than expectorate or saliva, substances such as proteins, polysaccharides and lipids eluting with the nucleic acid from the cells partially or completely inhibit the nucleic acid amplification reaction, and therefore non-reproducible results are obtained or the presence or level of expression of the target nucleic acid cannot be accurately evaluated.
In addition, the surfactant used in preparing the nucleic acid amplification sample from the biological sample can partially or completely inhibit the nucleic acid amplification reaction if it is present above a certain concentration in the nucleic acid amplification sample, and therefore the nucleic acid amplification sample must be diluted for the nucleic acid amplification reaction.
For these reasons it has been very difficult to perform diagnosis of patients by amplification of target nucleic acid and to make accurate tests for foods or environmental sanitation, or of animals or plants.
[Patent document 1] Japanese Unexamined Patent Publication HEI No. 9-173065